Abstract

Herbaspirillum seropedicae is a diazotrophic Gram-negative bacterium, originally isolated as endophyte from different plants, including rice and sugar cane. Strains Z69, Z78 and Z67 are closely related and accumulate significant amounts of poly-3-hydroxybutyrate (P3HB) when are cultured on glucose or other carbohydrates as sole carbon sources. H. seropedicae Z69 is able to synthesize the copolymer of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] when consumes propionic acid alone or mixed with glucose or xylose as carbon sources. When a carbohydrate and this organic acid are consumed together, the sugar is used for bacterial growth and 3HB synthesis, while propionic acid is partially used as precursor for the synthesis of 3-hydroxyvalerate (3HV). According to KEGG database, the catabolism of propionic acid by H. seropedicae follows the routes of 2-methylcitrate (prpC gene) and 3-methyl-malonyl-CoA ( mmsA gene). The objective of this work is to remove these catabolic pathways, redirecting the molecules of propionic acid to the synthesis of 3HV (Y3HV/prp, grams of 3HV produced per gram of propionic acid consumed). By cross-over PCR, we constructed two mutant strains derived from H. seropedicae Z69 (wt): Z69prpC (prpC, methyl citrate synthase) and Z69mmsA (mmsA, methylmalonate-semialdehyde dehydrogenase). The growth of these strains was evaluated using different concentrations (0.5, 1, 2 y 3 g/l) of propionate as sole carbon source. Z69prpC was unable to grow in any condition, but Z69mmsA and Z69 (wt) exhibited a comparable growth profile. Y3HV/prp was determined in strains Z69prpC and Z69 (wt) grown in batch cultures with a mixture of glucose and propionate as carbon sources. Y3HV/prp of Z69prpC was about 0.23 g/g, about eight times higher than the yield reached by strain Z69 (wt) (0.03 g/g). According with these results, the methylcitrato pathway is required by H. seropedicae Z69 for propionic acid catabolism. The mutant Z69prpC, unable to metabolize propionyl-CoA, showed a higher synthesis of 3HV (expressed as percentage of 3HV per gram of biomass) and a higher percentage (w/w) of this monomer in the copolymer accumulated, P(3HB-co-3HV). New strategies for growing the mutant strain Z69prpC using propionate and glucose as carbon sources are being assayed to increase the yield of 3HV from propionate (Y3HV/prp).